Method of Treating Neurological Conditions with Oleandrin

ABSTRACT

A method of treating neurological condition in a subject by administration of a cardiac glycoside is provided. Alzheimer&#39;s disease, Huntington&#39;s disease or stroke are treated by administering a therapeutically effective amount of cardiac glycoside to a subject. The cardiac glycoside can be present in a dosage form.

CROSS-REFERENCE TO EARLIER FILED APPLICATION

The present application claims the benefit of U.S. ProvisionalApplication No. 61/293,812 filed Jan. 11, 2010, and is a continuation ofU.S. Ser. No. 12/987,693 filed Jan. 10, 2011, the entire disclosures ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns a method of treating neurologicalconditions with cardiac glycoside compounds or preparations containingthem. In particular, the invention concerns a method for treatingneurological disease or disorder by administration of a cardiacglycoside to a subject in need thereof.

BACKGROUND OF THE INVENTION

Neurological diseases and disorders affect brain function. Many effortshave been made to develop curative or ameliorative therapies for thesediseases and disorders; however, no comprehensive or universallycurative therapy has been developed, even though there are numerouspharmacotherapeutic approaches that have been proven to be effectiveagainst various different diseases and disorders.

Huntington's disease (HD) is an inherited disease of the brain thataffects the nervous system. It is caused by a defective gene that ispassed from parent to child. The HD gene interferes with the manufactureof a particular protein known as ‘huntington’ which appears to becrucial for proper brain development. The classic signs of HD includeemotional, cognitive and motor disturbances. Huntington's ischaracterized by jerky involuntary movements (chorea), but sometimescauses rigidity without abnormal movements, changes in using the limbs(apraxia), loss of control of bodily functions and dementia, including aprogressive deterioration of memory, speed of thought, judgment, andlack of awareness of problems and planning. There is no known cure forHuntington's disease. Although there are a number of medications to helpcontrol symptoms associated with HD such as emotional and movementproblems, there is no treatment to stop or reverse the course of thedisease. Huntington's disease has been recognized as a disease with ageneral membrane abnormality. A significantly elevated level andactivity (10 fold increase) of Na,K-ATPase has been observed inmembranes of erythrocytes and basal ganglia of Huntington's patientscompared to that of normal (Butterfield D A, Oeswein J Q, Prunty M E,Hisle K C, Markesbery W R). Increased sodium, potassium adenosinetriphosphatase activity in erythrocyte membranes in Huntington'sdisease. Ann Neurology, 4:60-62, 1978) fibroblast membranes obtainedfrom the skin of Huntington's disease patients (Schroeder F, Goetz I E,Roberts E, Membrane anomalies in Huntington's disease fibroblasts. J.Neurochem. 43: 526-539, 1984).

Alzheimer's disease is a form of dementia—a neurodegenerative diseasethat damages the brain's intellectual functions (memory, orientation,calculation, etc.), but usually preserves its motor functions. InAlzheimer's disease, the mind gradually deteriorates, causing memoryloss, confusion, disorientation, impaired judgment and other problemsthat may affect a person's ability to perform normal daily activities.The type, severity, sequence and progression of mental changes varygreatly. There is no known cure for Alzheimer's disease and no known wayto slow its progression. For some people in the early or middle stagesof the disease, medication such as tacrine may alleviate some cognitivesymptoms. Aricept (donepezil) and Exelon (rivastigmine) are reversibleacetylcholinesterase inhibitors that are indicated for the treatment ofmild to moderate dementia of the Alzheimer's type. These drugs (calledcholinesterase inhibitors) work by increasing the brain's levels of theneurotransmitter acetylcholine, helping to restore communication betweenbrain cells. Some medications may help control behavioral symptoms suchas sleeplessness, agitation, wandering, anxiety, and depression. Thesetreatments are aimed at making the patient more comfortable. Although nomedication is known to cure Alzheimer's disease, cholinesteraseinhibitors may improve performance of daily activities, or lessenbehavioral problems. Medications for the treatment of Alzheimer'sdisease currently being tested include oestrogens, nonsteroidalanti-inflammatory agents, vitamin E, selegiline (Carbex, Eldepryl) andthe botanical product gingko biloba.

Under normal conditions, neurons maintain their testing membranepotential and function regulated by membrane-bound, homeostatic,energy-dependent Na-K-ATPase pumps. Ischemia triggers alterations in ionhomeostatsis potentially leading to irreversible tissue injuries.Compromised Na,K-ATPase activity has been suggested to play a role in aneuropathologic and apoptotic process in some models of focal ischemiaand traumatic brain injury. The role of Na,K-ATPase in stroke-mediatedischemic brain injury has been reported to be associated with severaldifferent molecular mechanisms. Inhibition of Na,K-ATPase catalyticactivity can, for example, lead to a reduction of ATP consumption duringischemia-reperfusion. Additionally, the deletion of cytosolic Ca²⁺ maycause neuronal cell death. Thus, inhibition of Na,K-ATPase, such as bycardiac glycosides, may result in an increase in intracellular Ca²⁺levels and a decline in extrusion of intracellular Ca²⁺ via the Na—Caexchanger. In line with this, the relatively lower levels ofintracellular Ca²⁺ in hippocampal CA1 neurons has been observed threedays after transient ischemia and elevation of calcium levels isbelieved to provide protection against delayed neuronal death across awide range of post-ischemic treatment times.

One of the pharmacological mechanisms of action of cardiac glycosidesinvolves their ability to bind to the ion exchange pump, Na, K-ATPaseand to inhibit the activity of this particular enzyme. Na, K-ATPase, thetransmembrane protein that catalyzes the active transport of Na⁺ and K⁺across the plasma membrane, is a well established pharmacologic receptorfor cardiac glycosides. This enzyme hydrolyzes ATP and uses the freeenergy to drive transport of K⁺ into the cell and Na⁺ out of cells,against their electrochemical gradients (Hauptman, P. J., Garg, R., andKelly, R. A. Cardiac glycosides in the next millennium. Prog.Cardiovasc. Dis. 41: 247-254, 1999).

Na, K-ATPase is composed of two heterodimer subunits, the catalyticα-subunit and the glycosylated β-subunit. There is also a γ subunit, butit has not been studied in detail. The α-subunit has binding sites forATP, Na⁺, K⁺, and cardiac glycosides. The β-subunit functions tostabilize the catalytic α-subunit and may play a regulatory role aswell. Four different a isoforms (α1, α2, α3, α4) and three different βisoforms (β1, β2, and β3) have been identified in mammalian cells. Therelative expression of each subunit type is markedly altered in normaland diseased states. Additionally, the apparent affinity of cardiacglycosides to the different a isoforms is quite different. Binding ofcardiac glycosides to the α1 isoform is less than that which occurs withthe α2 and α3 isoforms which are 250-fold or higher more sensitive toinhibition by this type of drug (Blanco, G. and Mercer, R. W. Isozymesof the Na, K-ATPase: heterogeneity in structure, diversity in function.Am. J. Physiol. 275 (Renal Physiol. 44): F633-F650, 1998). Sakai et al.(FEBS Letters 563: 151-154, 2004) report that expression of the α3subunit isoform is increased in human colorectal cancer cells ascompared to normal colorectal cells.

There is a broad range in relative water as opposed to lipid solubilityof cardiac glycosides. While most cardiac glycosides can bind to andinhibit Na,K-ATPase activity, those cardiac glycosides which arerelatively more water soluble (hydrophilic) than lipid soluble(lipophilic or hydrophobic) have only a limited ability to cross thelipid barrier to the brain known as the blood-brain bather. Theblood-brain barrier (BBB) is a separation of circulating blood andcerebrospinal fluid (CSF) maintained by the choroid plexus in thecentral nervous system (CNS). Endothelial cells restrict the diffusionof microscopic objects (e.g. bacteria) and large or hydrophilicmolecules into the CSF, while allowing the diffusion of smallhydrophobic molecules (O₂, hormones, lipid soluble cardiac glycosides,etc)

Nerium oleander is an ornamental plant widely distributed in subtropicalAsia, the southwestern United States, and the Mediterranean. Its medicaland toxicological properties have long been recognized. It has beenused, for example, in the treatment of hemorrhoids, ulcers, leprosy,snake bites, and even in the induction of abortion. Oleandrin, animportant component but not the sole component of oleander extract, is acardiac glycoside.

Extraction of glycosides from plants of Nerium species has providedpharmacologically/therapeutically active ingredients from Neriumoleander. Among these are oleandrin, neriifolin, and other cardiacglycoside compounds. Oleandrin extracts obtained by hot-water extractionof Nerium oleander, sold under the trademark ANVIRZEL™, contain theconcentrated form or powdered form of a hot-water extract of Neriumoleander. A Phase I trial of a hot water oleander extract (i.e.Anvirzel™) has been completed (Mekhail et al., Am. Soc. Clin. Oncol.,vol. 20, p. 82b, 2001). It was concluded that oleander extracts, whichwould provide about 57 ug oleandrin/day, can be safely administered atdoses up to 1.2 mL/m²/d. No dose limiting toxicities were found.

Huachansu is an extract obtained from toad skin and it comprisesbufadienolides, such as bufalin, a cardiac glycoside. HuaChanSu is anapproved medication for the treatment of cancer in China. It has beenused to treat various cancers, including hepatic, gastric, lung, skin,and esophageal cancers.

SUMMARY OF THE INVENTION

The invention provides a method of treating a neurological conditioncomprising administering to a subject in need thereof a compositioncontaining a cardiac glycoside in an effective amount to treat saidneurological condition.

Another aspect of the invention provides a method of treating, in asubject in need thereof, a neurological disease or disorder having anetiology associated with altered Na,K-ATPase activity with a compositioncomprising cardiac glycoside, the method comprising:

-   determining that the subject has a neurological disease or disorder    having an etiology associated with altered Na,K-ATPase α3 isoform to    α1 isoform subunit ratio or associated with altered Na,K-ATPase    activity; and-   indicating administration to the subject a composition comprising    cardiac glycoside.

Some embodiments of the invention include those wherein: 1) the subjectis prescribed and administered a therapeutically relevant dose ofcomposition comprising cardiac glycoside; 2) the subject is administeredthe composition comprising cardiac glycoside according to a prescribeddosing regimen; 3) the subject is administered a composition comprisingan extract comprising a cardiac glycoside; 4) the extract furthercomprises one or more other therapeutically effective agents obtainedalong with the cardiac glycoside during extraction; 5) the extractfurther comprises one or more other therapeutically effective agentsobtained along with the cardiac glycoside during extraction; 6) thecomposition further comprises one or more other non-cardiac glycosidetherapeutically effective agents, i.e. an agent that is not a cardiacglycoside; and/or 7) the subject is administered a hot water extract ofa plant or animal source containing cardiac glycosides 2 mg to 22.5 mgper day, or a concentrated extract (e.g. supercritical CO₂ extract ororganic solvent extract) of a plant or animal source of cardiacglycosides ranging from 0.6 to 4.8 mg, or a pure single chemical form ofa cardiac glycoside ranging from 10 to 500 μg.

The invention also provides a method of treating a neurologicalcondition in a subject in need thereof comprising:

-   determining whether or not the neurological condition in the subject    is Alzheimer's disease, Huntington's disease, stroke or other    neurological condition;-   indicating administration of cardiac glycoside;-   administering an initial dose of cardiac glycoside to the subject    according to a prescribed initial dosing regimen for a period of    time;-   periodically determining the adequacy of subject's clinical response    and/or therapeutic response to treatment with cardiac glycoside; and-   if the subject's clinical response and/or therapeutic response is    adequate, then continuing treatment with cardiac glycoside as needed    until the desired clinical endpoint is achieved; or-   if the subject's clinical response and/or therapeutic response are    inadequate at the initial dose and initial dosing regimen, then    escalating or deescalating the dose until the desired clinical    response and/or therapeutic response in the subject is achieved.

The invention also provides a method of preventing or reducing theincidence of occurrence of a neurological condition in a population ofsubjects at risk thereof, the method comprising:

-   administering an effective dose of cardiac glycoside on a recurring    basis for an extended period of time to one or more subjects in a    population of subjects at risk of suffering from a neurological    condition such as Alzheimer's disease, Huntington's disease, stroke    or other neurological condition, thereby preventing or reducing the    incidence of the neurological condition in the population.

The invention also includes embodiments wherein: a) the method furthercomprises indicating administration of cardiac glycoside to the one ormore subjects; b) the method further comprises administering aneffective dose of cardiac glycoside to the subject according to aprescribed dosing regimen for a period of time; c) the method furthercomprises periodically determining the adequacy of one or more subject'sclinical response and/or therapeutic response to treatment with cardiacglycoside; d) if the subject's clinical response and/or therapeuticresponse is adequate, then the method further comprises continuingtreatment with cardiac glycoside as needed until the desired clinicalendpoint is achieved; e) if the subject's clinical response and/ortherapeutic response are inadequate at the initial dose and initialdosing regimen, then the method further comprises escalating ordeescalating the dose until the desired clinical response and/ortherapeutic response in the subject is achieved; f) the cardiacglycoside is administered to plural subjects in a population; g) therecurring basis is daily, every other day, every second day, every thirdday, every fourth day, every fifth day, every sixth day, weekly, everyother week, every second week, every third week, monthly, bimonthly,semi-monthly, every other month every second month, quarterly, everyother quarter, trimesterly, seasonally, semi-annually and/or annually;h) the extended period is one or more weeks, one or more months, one ormore quarters and/or one or more years; i) the effective dose isadministered one or more times in a day; j) the method further comprisesidentifying a population of subjects at risk of suffering from aneurological condition such as Alzheimer's disease, Huntington'sdisease, stroke or other neurological condition; and/or k) thepopulation of subjects at risk is characterized by advancing age of thesubject, familial history of the neurological condition, geneticpredisposition to occurrence of neurological condition, the presence andexpression of ApoE4 gene in the subject, female gender (twice as manywomen get Alzheimer's disease than men), cardiovascular disease (e.g.high blood pressure and high cholesterol levels), diabetes (especiallyType 2 or adult onset forms of this disease), Down's Syndrome, headinjury, low levels of formal education, smoking, excessive alcoholconsumption and/or drug abuse.

The invention also provides a time-delayed method of treating stroke ina subject comprising:

-   within an acceptable delay period after a subject has suffered the    stroke, administering an initial dose of cardiac glycoside according    to an initial dosing regimen;-   determining the adequacy of subject's clinical response and/or    therapeutic response to treatment with cardiac glycoside; and-   if the subject's clinical response and/or therapeutic response is    adequate, then continuing treatment with cardiac glycoside as needed    until the desired clinical endpoint is achieved; or-   if the subject's clinical response and/or therapeutic response are    inadequate at the initial dose and initial dosing regimen, then    escalating or deescalating the dose until the desired clinical    response and/or therapeutic response in the subject is achieved.

Some embodiments of the invention include those wherein: 1) the delayperiod is 10 hours or less, 8 hours or less, 6 hours or less, 4 hours orless, 3 hours or less, 2 hours or less, 1 hour or less, 45 minutes orless, 30 minutes or less, 20 minutes or less or 10 min or less; 2)determining the adequacy of a subject's clinical and/or therapeuticresponse is done by assessments of any weakness of the face, arm and/orleg on one side of the body, numbness in the face, arm, and/or leg onone side of the body, inability to understand spoken language, inabilityto speak or speak clearly, inability to write, vertigo and/or gaitimbalance, double vision and an unusually severe headache; or 3) acombination thereof.

The invention also provides use of a cardiac glycoside in themanufacture of a medicament for the treatment of a neurologicalcondition in a subject. In some embodiments, the manufacture of such amedicament comprises: providing a cardiac glycoside; including a dose ofcardiac glycoside in a pharmaceutical dosage form; and packaging thepharmaceutical dosage form. In some embodiments, the manufacture can beconducted as described in PCT International Application No.PCT/US06/29061. The manufacture can also include one or more additionalsteps such as: delivering the packaged dosage form to a vendor(retailer, wholesaler and/or distributor); selling or otherwiseproviding the packaged dosage form to a subject having a neurologicalcondition; including with the medicament a label and a package insert,which provides instructions on use, dosing regimen, administration,content and toxicology profile of the dosage form. In some embodiments,the treatment of a neurological condition comprises: determining that asubject has a neurological disease or disorder; indicatingadministration of cardiac glycoside to the subject according to a dosingregimen; administering to the subject one or more pharmaceutical dosageforms containing cardiac glycoside, wherein the one or morepharmaceutical dosage forms is administered according to the dosingregimen.

The invention also provides a cardiac glycoside or cardiacglycoside-containing composition, i.e. a pharmaceutical formulation ordosage form, for the treatment of a neurological condition. In someembodiments, the cardiac glycoside-containing composition is asdescribed herein or in U.S. Pat. No. 7,402,325, PCT InternationalApplication No. PCT/US06/29061, U.S. application Ser. No. 12/019,435,the entire disclosures of which are hereby incorporated by reference.

In some embodiments, the subject having a neurological condition, i.e.the subject in need thereof, is part of a population of such subjects.The invention provides a method for improving the clinical status of astatistically significant number of subjects of in a population ofsubjects having a neurological condition, the method comprising:administering to the population of subjects a cardiac glycoside orcardiac glycoside-containing composition; and determining the clinicalstatus of the subjects. In some embodiments, the statisticallysignificant number is at least 5% of the population.

In some embodiments, the neurological condition is Alzheimer's disease,Huntington's disease, stroke or other neurological condition, such asdescribed herein. The medicament can be manufactured by inclusion of thecardiac glycoside in a pharmaceutical dosage form containing one or morepharmaceutically acceptable excipients.

Treatment of the subject with cardiac glycoside is continued as needed.The dose or dosing regimen can be adjusted as needed until the patientreaches the desired clinical endpoint(s) such as a reduction oralleviation of specific neurological symptoms associated with thedisease. Determination of the adequacy of clinical response and/ortherapeutic response can be conducted by a clinician familiar with theneurological condition being treated.

In some embodiments, the neurological condition is selected from thegroup consisting of neurological disease, neurological disorder, andstroke. In some embodiments, the neurological disease is aneurodegenerative disease. In some embodiments, the neurodegenerativedisease is selected from the group consisting of Huntington's disease,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,bovine spongiform encephalopathy, multiple sclerosis, diabeticneuropathy, autism and juvenile neuronal ceroid lipofuscinosis. In someembodiments, stroke is ischemic stroke or stroke-mediated ischemicinjury.

In some embodiments: 1) the cardiac glycoside is selected from the groupconsisting of oleandrin, ouabain, bufalin, digitoxin, cinobufatalin,cinobufagin, and resibufogenin; 2) the cardiac glycoside is present inpure form whether derived through extraction of a plant or animalsource, synthesized or manufactured through chemical modification (e.g.derivatization) of an available cardiac glycoside; 3) the cardiacglycoside is present in an extract; 4) the cardiac glycoside is presentin a pharmaceutical formulation or composition; 5) the cardiac glycosidehas been obtained from an oleander plant mass; 6) the oleander plantmass comprises Nerium species, such as Nerium oleander, or of Thevetiaspecies, such as Thevetia neriifolia (otherwise known as yellowoleander); 7) the cardiac glycoside crosses the blood brain barrier(BBB) after administration to the subject; 8) the cardiac glycosideextract was prepared by supercritical fluid (SCF) extraction optionallyin the presence of a modifier; 9) the cardiac glycoside is oleandrin;10) the cardiac glycoside has a clearance rate for brain tissue of nogreater than 4 L/hr; and/or 11) following administration thereof by anyof the routes disclosed herein, the cardiac glycoside is retained in thebrain tissue for a period of at least 8 hours. In some embodiments, thecardiac glycoside excludes neriifolin.

In some embodiments: 1) the SCF extract further comprises at least oneother pharmacologically active agent aside from the cardiac glycoside,said other pharmacologically active agent having been obtained alongwith the cardiac glycoside during the extraction process used to preparethe extract; 2) the SCF extract further comprises at least one othernon-cardiac glycoside pharmacologically active agent; 3) the otherpharmacologically active agent may contribute to the therapeuticefficacy of the cardiac glycoside when the extract is administered to asubject; 4) the other pharmacologically active agent functionsadditively or synergistically to contribute to the therapeutic efficacyof the cardiac glycoside; and/or 5) the extract has been obtained fromtoad skin or secretions therefrom.

In some embodiments, the neurological condition has an etiologyassociated with specific changes in Na,K-ATPase activity either throughincreased enzyme activity such as that associated with juvenile autism(Clin. Biochem. 2009; 42 (10-11), 949-957), decreased enzyme activitysuch as that observed in diabetic neuropathy (Neuroscience 2009), Battendisease (Exp. Cell Res. 2008, 314(15):2895-2905), age-related dementiaand Alzheimer's disease (J. Alzheimers Dis. 2008, 14(1): 85-93;Neurobiol. Aging 2007, 28(7):987-994), or specific mutations inNa,K-ATPase subunit structure such as that associated with Parkinson'sdisease (Human Gen. 2009, 126(3):431-447).

The individual steps of the methods of the invention can be conducted atseparate facilities or within the same facility.

In some embodiments, the neurons are in vitro, ex vivo or in vivo. Insome embodiments, the neurons are CA-1 neurons.

The invention also provides: a method of treating a neurologicalcondition; a method of treating, in a subject in need thereof, aneurological disease or disorder having an etiology associated withaltered Na,K-ATPase activity; use of a cardiac glycoside in themanufacture of a medicament for the treatment of a neurologicalcondition; and/or a cardiac glycoside or cardiac glycoside-containingcomposition for the treatment of a neurological condition substantiallyas shown and described herein.

The invention includes all combinations of the aspects, embodiments andsub-embodiments of the invention disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present description and describeexemplary embodiments of the claimed invention. The skilled artisanwill, in light of these figures and the description herein, be able topractice the invention without undue experimentation.

FIGS. 1A-1C depict results of the comparative evaluation of oleandrinversus neriifolin in a neuroprotection brain-slice-based “stroke” assay(Example 8), wherein the number of healthy cortical neurons isdetermined following 5-6 minutes of oxygen and glucose deprivation (OGD)in the presence or absence of those agents. The results of threeindependent experiments are presented.

FIG. 1D depicts concentration-response data obtained from thecomparative evaluation of the oleandrin versus neriifolin, the control,in a neuroprotection brain-slice-based “stroke” assay (Example 11),wherein the number of healthy cortical neurons is determined following5-6 minutes of oxygen and glucose deprivation (OGD) in the presence orabsence of those agents.

FIG. 1E depicts results of a concentration-response assay for anoleandrin-containing SCF extract in a neuroprotection brain-slice-based“stroke” assay as described herein (Example 11), wherein no oxygen orglucose deprivation is used as control.

FIGS. 2A-2C depict results of the comparative evaluation of oleandrinversus neriifolin in a neuroprotection brain-slice-based “non-stroke”assay (Example 8), wherein the number of healthy cortical neurons isdetermined without OGD in the presence or absence of those agents.

FIGS. 3A-3C depict results of the comparative evaluation of oleandrinversus oleandrin extract in a neuroprotection brain-slice-based“Alzheimer's” assay (Example 9), wherein the number of healthy corticalneurons is determined following APP/Aβ-induced degeneration in theabsence or presence of varying amount of those agents.

FIGS. 4A-4D depict results from duplicate experiments of the comparativeevaluation of oleandrin in a neuroprotection cortico-striatal co-cultureneuron-based “Huntington's disease” assay (Example 10), wherein thepercent rescue, relative to control, of cortical neurons and striatalneurons transfected with a mutant form of the Huntington (htt) proteinis determined in the absence or presence of varying amount of oleandrin.

FIG. 4E depicts the concentration-response curve for oleandrin in termsof its relative ability to rescue neuronal injury and death due totransfection of Huntington's disease (results from in vitro assay;Example 10).

FIG. 5 depicts the results of a time-delayed neuroprotectionbrain-slice-based “stroke” assay as described herein (Example 13),wherein the “stroked” brain tissue is treated with cardiacglycoside-containing solution (oleandrin-containing SCF extract) afterexpiration of about 2, about 4 or about 6 hours following OGD.

FIG. 6 depicts a plot of the concentration-time relation in plasma andbrain tissue for oleandrin over a 24-hour period following i.p.administration of an oleandrin-containing SCF extract (triangles,identifier code: PBI-05204) and oleandrin (circles) in CD1 mice. Mean±SDoleandrin concentration in brain (ng/g) and plasma (ng/ml) for 5 miceare shown. PBI-05204 (38 mg/kg containing 0.8 mg/kg oleandrin, 50 μL)and oleandrin (3 mg/kg, 100 μL) were dissolved in DMSO:PEG400 vehicle,50:50 v/v (PBI-05204, 28.6 mg/ml) or 25:75 v/v (oleandrin, 1 mg/ml).Control groups received DMSO:PEG400 vehicle alone. At 0.5, 1, 2, 4, 8and 24 hours, plasma and brain tissue were harvested for LC/MS/MSanalysis. Rapid and sustained CNS penetration was observed followingboth PBI-05204 and oleandrin administration.

FIGS. 7A and 7B depict the results of a neuroprotection brain-slicebased assay conducted as described herein. FIG. 7A depicts aconcentration-response curve showing the relation for oleandrin (grayfilled circles). A compendium of 6 runs normalized to the differencebetween internal positive (non-OGD) and negative (OGD) controls for eachrun set to 100%; means±SEM are shown. The concentration relation forneriifolin (open squares) is reprinted here as taken from Wang et al.(Proc. Natl. Acad. Sci. USA (2006), 103(27), 10461-10466) forcomparison. FIG. 7B depicts a concentration response curve showing therelation for PBI-05204 (black filled squares). A compendium of 13 runsnormalized to the difference between internal positive (non-OGD) andnegative (OGD) controls for each run set to 100%; means±SEM are shown.Data from FIG. 7A for oleandrin are replotted in FIG. 7B assuming a 3%composition for oleandrin in the PBI-05204 extract.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of treating a neurological condition byadministration of an effective dose of cardiac glycoside to a subject inneed thereof. The cardiac glycoside is administered according to adosing regimen best suited for the subject, the suitability of the doseand dosing regimen to be determined clinically according to conventionalclinical practices and clinical treatment endpoints for the neurologicalcondition being treated.

In some embodiments, the neurodegenerative disorder or neurologicalcondition being treated has an etiology associated with Na,K-ATPasebinding activity or with disregulation of Na,K-ATPase within a cell ortissue. In some embodiments, the neurodegenerative disorder orneurological condition being treated has an etiology associated withHIF-1α binding activity or with disregulation of HIF-1α within a cell ortissue. Such disregulation may occur, for example, as a significantalteration in the extent of enzymatic activity. This, in turn, mayresult from a change in enzyme function, enzyme content or evendistribution within an affected tissue.

A subject treated according to the invention will exhibit a therapeuticresponse. By “therapeutic response” is meant that a subject sufferingfrom the disease or disorder will enjoy at least one of the followingclinical benefits as a result of treatment with a cardiac glycoside:amelioration of the disease or disorder, reduction in the occurrence ofsymptoms associated with the disease or disorder, partial remission ofthe disease or disorder, full remission of the disease or disorder, orincreased time to progression. In other words, the therapeutic responsecan be a full or partial therapeutic response.

A therapeutic response can also be described as one in which the qualityof life of the patient afflicted with the neurodegenerative disease isimproved Improvement in quality of life may occur, for example, througha reduction in occurrence, frequency or severity of symptoms associatedwith the disease (e.g. tremors, involuntary muscle movements, loss orpartial loss of nerve-muscle coordination, memory retention, etc.).

“Preventing occurrence of a neurological condition in a population ofsubjects at risk” means that the neurological condition will not occurduring a predetermined time period in a demographically predeterminedpopulation of subjects that are at risk of suffering from theneurological condition. The prevention during the predetermined timeperiod occurs as a result of subjects in that population having beenadministered a cardiac glycoside according to the methods of theinvention. As one example, when a cardiac glycoside-containingcomposition is administered for a predetermined time period to subjectsin a population of subjects at risk of suffering from stroke, strokewill not occur in those subjects during the predetermined time period.In particular, a cardiac glycoside-containing composition is chronicallyadministered over a period of one year to a population of subjects atrisk of suffering from Alzheimer's disease, and the subjects in thatpopulation do not exhibit symptoms associated with Alzheimer's duringthat one-year period.

“Reducing the incidence of occurrence of a neurological condition in apopulation of subjects at risk” is related in meaning to “preventing theincidence”, except that “reducing the incidence of occurrence” permitsthe occurrence of the neurological condition in a demographicallypredetermined population of subjects but at a rate of occurrence or alevel of severity that is reduced as compared to an otherwisedemographically similar predetermined population of subjects at risk notbeing administered the cardiac glycoside-containing compositionaccording to the methods of the invention.

As used herein, “time to progression” is the period, length or durationof time after a disease is diagnosed (or treated) until the diseasebegins to worsen. It is the period of time during which the level of adisease is maintained without further progression of the disease, andthe period of time ends when the disease begins to progress again.Progression of a disease is determined by “staging” a subject sufferingfrom a neurological condition prior to or at initiation of therapy. Forexample, the subject's neurological health is determined prior to or atinitiation of therapy. The subject is then treated with cardiacglycoside, and the neurological health monitored periodically. At somelater point in time, the symptoms of the neurological condition mayworsen, thus marking progression of the disease and the end of the “timeto progression”. The period of time during which the disease did notprogress or during which the level or severity of the disease did notworsen is the “time to progression”.

A dosing regimen includes a therapeutically relevant dose (or effectivedose) of one or more cardiac glycosides administered according to adosing schedule. A therapeutically relevant dose, therefore, is atherapeutic dose at which a therapeutic response of the disease ordisorder to treatment with a cardiac glycoside is observed and at whicha subject can be administered the cardiac glycoside without an excessiveamount of unwanted or deleterious side effects. A therapeuticallyrelevant dose is non-lethal to a subject, even though it may cause someside effects in the patient. It is a dose at which the level of clinicalbenefit to a subject being administered the cardiac glycoside exceedsthe level of deleterious side effects experienced by the subject due toadministration of the cardiac glycoside. A therapeutically relevant dosewill vary from subject to subject according to a variety of establishedpharmacologic, pharmacodynamic and pharmacokinetic principles. However,a therapeutically relevant dose (relative, for example, to oleandrin)will typically not exceed 25, 100, 250, 500 or 1000 micrograms ofcardiac glycoside/day or it can be in the range of 25-500 or 25-1000micrograms of cardiac glycoside/day. It is known in the art that theactual amount of a drug required to provide a target therapeutic resultin a subject may vary from subject to subject according to the basicprinciples of pharmacy.

A therapeutically relevant dose can be administered according to anydosing regimen typically used in the treatment of neurological orneurodegenerative diseases or disorders. A therapeutically relevant dosecan be administered once, twice, thrice or more daily dosing schedule.It can be administered every other day, every third day, every fourthday, every fifth day, semiweekly, weekly, biweekly, every three weeks,every four weeks, monthly, bimonthly, semimonthly, every three months,every four months, semiannually, annually, or according to a combinationof any of the above to arrive at a suitable dosing schedule. Forexample, a therapeutically relevant dose can be administered once dailyfor one or more weeks.

The examples below include evidence of the efficacy of cardiacglycosides in neurological conditions such as neurological diseases,neurological disorders and stroke. Example 3 details a method oftreating Alzheimer's disease with cardiac glycoside or a combination ofcardiac glycoside with one or more other therapeutic agents. Example 4details a method of treating Huntington's disease with cardiac glycosideor a combination of cardiac glycoside with one or more other therapeuticagents. Example 5 details a method of treating stroke-mediated ischemicbrain injury with cardiac glycoside or a combination of cardiacglycoside with one or more other therapeutic agents.

In general, a subject having a neurological condition is treated asfollows. A subject presenting with a neurological condition is evaluatedto determine whether or not the neurological condition is Alzheimer'sdisease, Huntington's disease, stroke or other neurological condition.Administration of cardiac glycoside is indicated. Initial doses ofcardiac glycoside are administered to the subject according to aprescribed dosing regimen for a period of time. The subject's clinicalresponse and level of therapeutic response are determined periodically.If the level of therapeutic response is too low at one dose, then thedose is escalated according to a predetermine dose escalation scheduleuntil the desired level of therapeutic response in the subject isachieved. Treatment of the subject with cardiac glycoside is continuedas needed. The dose or dosing regimen can be adjusted as needed untilthe patient reaches the desired clinical endpoint(s) such as cessationof the disease itself, reduction in disease associated symptoms, and/ora reduction in the progression of the disease process.

Example 8 provides a detailed description of an in vitro assay used toevaluate the efficacy of the cardiac glycosides for the treatment ofstroke-mediated ischemic neuronal injury. The assay is a brainslice-based assay for oxygen and glucose deprivation (OGD) used toinduce ≧50% loss of healthy cortical neurons by 24 hours. The cardiacglycoside Neriifolin (3 μM) is used as a positive control. Oleandrin wastested in OGD treated brain slices (stroke model, FIGS. 1A-1C) andnon-OGD treated brain slices (non-stroke model, FIGS. 2A-2C). The dataindicate that oleandrin provides substantial neuroprotection when thebrain slices (neurons) are exposed to solutions of oleandrin ranging inconcentration from 0.1 to 3 μM. While no direct measurements have beenmade in human brain following a systemic dose of oleandrin, it can beassumed from data obtained in a phase I study of oleander extract aswell as data previously obtained in rodent studies in which the abilityof oleandrin to specifically cross the blood brain barrier was examinedthat a dose level to reach this concentration of oleandrin in the humanbrain would be probably around 1-10 ng, about 3-6 ng, or about 4 ng.

Evidence of the existence of one or more pharmacologically activecomponents, other than oleandrin, in the SCF extract was obtained bycomparing the concentration-response curves for a solution containingpure oleandrin versus one containing the SCF extract. FIG. 1D depictsthe results of a concentration-response assay for an aqueous solutioncontaining pure oleandrin in a neuroprotection brain-slice-based“stroke” assay as described in Example 11. The concentration ofoleandrin in the aqueous solution was varied from 0.0069 to 230 μg/ml.FIG. 1E depicts results of a concentration-response assay for anoleandrin-containing SCF Nerium species extract in a neuroprotectionbrain-slice-based “stroke” assay as described herein (Example 11). Thedata demonstrate that the extract is more efficacious than pureoleandrin meaning the extract contains one or more pharmacologicallyactive agents that provide neuroprotection.

Example 11 provides a detailed description of an in vitro assay used toevaluate the efficacy of the extract, or composition thereof, for thetreatment of stroke-mediated ischemic neuronal injury. The assay is abrain slice-based assay for oxygen and glucose deprivation (OGD) used toinduce 50% loss of healthy cortical neurons by 24 hours. The parentunfractionated SCF extract of Nerium species, e.g. Nerium oleander, isused as a positive control.

Accordingly, the invention provides a method of protecting neuronsagainst loss of activity caused by oxygen depletion or oxygen-glucosedepletion by exposing the oxygen depleted and/or glucose-depletedneurons to an effective amount of oleandrin or oleandrin-containingextract to minimize loss of activity, reduce the rate of loss ofactivity, stop the loss of activity, slow down onset of loss ofactivity, and/or protect the function of neurons caused by exposing theoxygen depleted and/or glucose-depleted conditions.

Example 9 provides a detailed description of an in vitro assay used toevaluate the efficacy of the cardiac glycosides for the treatment ofAlzheimer's disease. The assay is a brain slice-based assay forAPP/Aβ-induced (APP: amyloid precursor protein) degeneration of corticalpyramidal neurons. Upon cleavage by a secretase enzyme, the APP isreduced to Aβ peptides which are believed to be a causative factor inbeta-amyloid plaque formation. Aβ proteins are associated withbeta-amyloid plaque formation and are believed to be a hallmark if notetiologic factor in Alzheimer's disease. Biolistic transfection is usedto introduce vital markers such as YFP (a marker yellow fluorescentprotein) and to introduce disease gene constructs into the same neuronalpopulations in the brain slices. YFP is co-transfected with APP isoformsleading to the progressive degeneration of cortical pyramidal neuronsover the course of three to four days after brain slice preparation andtransfection. The data (FIGS. 3A-3C) indicate that oleandrin and anoleandrin-containing SCF extract provided a concentration-dependentneuroprotection to APP-transfected brain slices thereby rescuing levelsnearly to the same levels as provided by BACE inhibitor drugs, i.e. betasecretase inhibtor drugs. The beta secretase enzyme cleaves the APPprecursor protein into toxic Aβ-proteins. The oleandrin-containing SCFextract appeared to provide greater neuroprotection than oleandrinalone. The data in FIGS. 3A-3C are of significance in that few compoundsor therapeutic strategies in the literature have shown any significantprotection of neurons in this in vitro assay representative of Alzheimerdisease. The data indicate that cardiac glycosides such as oleandrinwill be effective as single agents, or in when present in extracts usedas single therapy or combined with other products such as BASEinhibitors for the treatment of Alzheimer's disease.

Accordingly, the invention provides a method of protecting neuronsagainst loss of activity caused by Alzheimer's disease, the methodcomprising: exposing the neurons exhibiting characteristics ofAlzheimer's disease to an effective amount of oleandrin oroleandrin-containing extract to minimize loss of activity, reduce therate of loss of activity, stop the loss of activity, slow down onset ofloss of activity, and/or critical functioning of the neurons caused byAlzheimer's disease.

Example 10 provides a detailed description of an assay used to evaluatethe efficacy of the cardiac glycosides for the treatment of Huntington'sdisease. Mutant htt protein is introduced via electroporation intohigh-density, mixed co-cultures of cortical neurons, striatal neurons,and glia. The striatal and cortical neurons are transfected withdifferent color fluorescent proteins thereby facilitating the separateidentification of the different types of neurons in the co-culture. Thecolor fluorescent proteins are fluorescent and ‘emit’ color uponactivation with a light source of appropriate wavelength. The data(FIGS. 4A-4D) indicate that oleandrin and oleandrin-containing SCFextract are more effective than KW6002 (an adenosine 2a receptorantagonist) in terms of providing a greater number of surviving neurons.Accordingly, the invention provides a method of protecting neuronsagainst loss of activity caused by Huntington's disease, the methodcomprising: exposing the neurons exhibiting characteristics ofHuntington's disease to an effective amount of oleandrin oroleandrin-containing extract to minimize loss of activity, reduce therate of loss of activity, stop the loss of activity, slow down onset ofloss of activity, and/or normal function of the neurons caused byHuntington's disease.

Example 13 details an exemplary brain-slice assay used as a model toevaluate the efficacy of cardiac glycoside in the treatment of stroke ina subject following completion of a delay period after the stroke. Thebrain-slice assay with oxygen glucose deprivation was conducted asdescribed herein; however, rather than treating the brain slicesprophylactically with cardiac glycoside, they were treated with thecardiac glycoside after delay periods of 0, 1, 2, 4, and 6 hours. Thedata, summarized in FIG. 5, demonstrates that cardiac glycoside, such asoleandrin or an SCF extract containing oleandrin, is effective atproviding significant neuroprotection for delay periods of up to 1, upto 2, up to 3, up to 4, up to 5, up to about 6 hours after the stroke.

Accordingly, the invention provides a time-delayed method of treatingstroke in a subject by administration of a dose of cardiac glycoside toa subject after the subject has suffered a stroke. Within an acceptabledelay period after a subject has suffered the stroke, an initial dose ofcardiac glycoside is administered according to an initial dosingregimen. Then, adequacy of the subject's clinical response and/ortherapeutic response to treatment with cardiac glycoside is determinedIf the subject's clinical response and/or therapeutic response isadequate, then treatment with cardiac glycoside is continued as neededuntil the desired clinical endpoint is achieved. Alternatively, if thesubject's clinical response and/or therapeutic response are inadequateat the initial dose and initial dosing regimen, the dose is escalated ordeescalated until the desired clinical response and/or therapeuticresponse in the subject is achieved. Dose escalation or de-escalationcan be performed in conjunction with a change in the dosing regimen,such as a change in dosing frequency or overall period of doseadministration.

Some of the brain slice assays herein are conducted under conditionswherein the brain tissue is treated with cardiac glycoside prior to OGD.Under those conditions, the data establishes the utility of cardiacglycosides at prophylactically providing neuroprotection against damagecaused by stroke.

If a clinician intends to treat a subject having a neurologicalcondition with a combination of a cardiac glycoside and one or moreother therapeutic agents, and it is known that the particularneurological condition, which the subject has, is at least partiallytherapeutically responsive to treatment with said one or more othertherapeutic agents, then the present method invention comprises:administering to the subject in need thereof a therapeutically relevantdose of cardiac glycoside and a therapeutically relevant dose of saidone or more other therapeutic agents, wherein the cardiac glycoside isadministered according to a first dosing regimen and the one or moreother therapeutic agents is administered according to a second dosingregimen. In some embodiments, the first and second dosing regimens arethe same. In some embodiments, the first and second dosing regimens aredifferent.

If the neurological condition being treated is Alzheimer's disease, theone or more other therapeutic agents can be selected from the groupconsisting of BACE inhibitors or acetylcholinesterase inhibitors. Insome embodiments, the one or more other therapeutic agents can beselected from the group consisting of Namenda™ (memantine HCl), Aricept™(donepezil), Razadyne™ (galantamine), Exelon™ (rivastigmine), andCognex™ (tacrine).

If the neurological condition being treated is Huntington's disease, theone or more other therapeutic agents can be selected from the groupconsisting of natural products, anticonvulsants, NMDA (n-methyld-aspartate) receptor antagonists, and sodium channel blockers.Exemplary agents include Vitamin E, Baclofen (a derivative of CoQ10),Lamotrigine (an anticonvulsant), remacemide (an anesthetic which is alow affinity NMDA antagonist), and riluzole (Na channel blocker). Theefficacy of each of these agents is considered to be low (Mestre T. etal, Chochrane Database Systematic Reviews Jul. 8, 2009; 8 (3): CD006455)on its own; however, it is expected that administration of a dosage formcontaining oleandrin or oleandrin-containing extract to subjectsreceiving one or more of these other agents will provide a subject,having a neurological disorder, an improved clinical affect as comparedto administration of these agents absent the oleandrin.

If the neurological condition being treated is stroke-mediated ischemicbrain injury (ischemic stroke), then the therapeutic treatmentsdisclosed in the literature (Gutierrez M. et al. “Cerebral protectiuon,brain repair, plasticity and cell therapy in ischemic stroke”Cerebrovasc. Dis. 2009; 27 Suppl 1:177-186), e.g. intravenousthrombolysis, can be employed in addition to the oleandrin or oleandrinextract-based method of the invention. In some embodiments, the one ormore other therapeutic agents can be selected from the group consistingof drugs such as Alteplase (a thrombolytic agent).

The one or more other therapeutic agents can be administered at dosesand according to dosing regimens that are clinician-recognized as beingtherapeutically effective or at doses that are clinician-recognized asbeing sub-therapeutically effective. The clinical benefit and/ortherapeutic effect provided by administration of a combination ofcardiac glycoside and one or more other therapeutic can be additive orsynergistic, such level of benefit or effect being determined bycomparison of administration of the combination to administration of theindividual cardiac glycoside and one or more other therapeutic agents.The one or more other therapeutic agents can be administered at dosesand according to dosing regimens as suggested or described by the Foodand Drug Administration, World Health Organization, European MedicinesAgency (E.M.E.A.), Therapeutic Goods Administration (TGA, Australia),Pan American Health Organization (PAHO), Medicines and Medical DevicesSafety Authority (Medsafe, New Zealand) or the various Ministries ofHealth worldwide.

The cardiac glycoside can be any cardiac glycoside known to possessNa,K-ATPase binding activity and/or HIF-1α (hypoxic inducing factoralpha) binding activity. The cardiac glycoside is capable of crossingthe blood-brain barrier and being retained in brain tissue for anextended period of time following administration. In this regard, thecardiac glycoside should be retained in the brain for at least 8 hoursfollowing administration of the cardiac glycoside due to tissue bindingand a consequent low clearance rate.

The cardiac glycoside can be present in pure form or as a mixture withone or more other compounds. The cardiac glycoside can be present as anextract. The extract can be prepared by supercritical fluid (SCF) carbondioxide (CO₂) extraction or a chemically modified form of such anextract (e.g. an extract that includes ethanol or was made using SCF CO₂and ethanol; Example 1). The extract can be obtained by extraction ofplant material with an organic solvent, e.g. ethanol, methanol, propanolor other such solvents. The extract can be obtained from plant or animalmaterial. The animal material can be the exudate of a toad (e.g. Bufobufo). The plant material can be plant mass such as obtained from Neriumspecies, such as Nerium oleander, or of Thevetia species, such asThevetia neriifolia or Thevetia puruviana (otherwise known as yellowoleander). The extraction process can be conducted on a dried powder ofNerium oleander leaves prepared according to a process described in acurrently-pending U.S. provisional application Ser. No. 60/653,210 filedFeb. 15, 2005 in the name of Addington or U.S. application Ser. No.11/340,016 filed Jan. 26, 2006 in the name of Addington, U.S.application Ser. No. 11/191,650 filed Jul. 28, 2006 (now U.S. Pat. No.7,402,325 issued Jul. 22, 2008) in the name of Addington, or PCTInternational Patent Application No. PCT/US06/29061 filed Jul. 26, 2006,the entire disclosures of which are hereby incorporated by reference, orby a process described herein.

As used herein, the term “oleandrin” is taken to mean all known forms ofoleandrin unless otherwise specified. Oleandrin can be present inracemic, optically pure or optically enriched form. Nerium oleanderplant material can be obtained, for example, from commercial plantsuppliers such as Aldridge Nursery, Atascosa, Tex.

Our work has shown that oleandrin has a good ability to cross the BBBand once in the brain tissues to reside there for a prolonged period oftime. That is, the residence time of oleandrin in the brain is longerthan that expected given the clearance of this molecule from plasma ofexperimental animals. As such, the relatively long residence time ofoleandrin in the brain provides a distinct advantage of this lipidsoluble cardiac glycoside compared to water soluble cardiac glycosidessuch as digoxin.

The extract can be obtained by modified (e.g. ethanol) or unmodifiedsupercritical fluid extraction of a cardiac glycoside-containing plantmass. The supercritical fluid extract can comprise at least one othercardiac glycoside pharmacologically active agent and/or at least oneother non-cardiac glycoside pharmacologically active agent thatcontributes to the therapeutic efficacy of the cardiac glycoside whenthe extract is administered to a subject. It can contribute additivelyor synergistically to the therapeutic efficacy of the cardiac glycoside.As used herein, the term “non-cardiac glycoside pharmacologically activeagent” (or component) means a compound that is not a cardiac glycoside.

The extract can be prepared by various different processes. The extractcan be prepared according to the process developed by Dr. Huseyin ZiyaOzel (U.S. Pat. No. 5,135,745) describes a procedure for the preparationof the extract of the plant in water. The aqueous extract reportedlycontains several polysaccharides with molecular weights varying from 2KD to 30 KD, oleandrin and oleandrigenin, odoroside and neritaloside.The polysaccharides reportedly include acidic homopolygalacturonans orarabinogalaturonans. U.S. Pat. No. 5,869,060 to Selvaraj et al.discloses hot water extracts of Nerium species and methods of productionthereof, e.g. Example 2. The resultant extract can then be lyophilizedto produce a powder. U.S. Pat. No. 6,565,897 (U.S. Pregrant PublicationNo. 20020114852 and PCT International Publication No. WO 2000/016793 toSelvaraj et al.) discloses a hot-water extraction process for thepreparation of a substantially sterile extract. Erdemoglu et al. (J.Ethnopharmacol. (2003) November 89(1), 123-129) discloses results forthe comparison of aqueous and ethanolic extracts of plants, includingNerium oleander, based upon their anti-nociceptive and anti-inflammatoryactivities. Organic solvent extracts of Nerium oleander are disclosed byAdome et al. (Afr. Health Sci. (2003) August 3(2), 77-86; ethanolicextract), el-Shazly et al. (J. Egypt Soc. Parasitol. (1996), August26(2), 461-473; ethanolic extract), Begum et al. (Phytochemistry (1999)February 50(3), 435-438; methanolic extract), Zia et al. (J.Ethnolpharmacol. (1995) November 49(1), 33-39; methanolic extract), andVlasenko et al. (Farmatsiia. (1972) September-October 21(5), 46-47;alcoholic extract). U.S. Pregrant Patent Application Publication No.20040247660 to Singh et al. discloses the preparation of a proteinstabilized liposomal formulation of oleandrin for use in the treatmentof cancer. U.S. Pregrant Patent Application Publication No. 20050026849to Singh et al. discloses a water soluble formulation of oleandrincontaining a cyclodextrin. U.S. Pregrant Patent Application PublicationNo. 20040082521 to Singh et al. discloses the preparation of proteinstabilized nanoparticle formulations of oleandrin from the hot-waterextract.

The SCF extraction can be conducted in the presence of a modifier in thesupercritical fluid, such as ethanol, to enhance extraction of thedesired compound(s) from the plant mass. Modifiers generally possessvolatility between that of the supercritical fluid and of the compoundbeing extracted, and they must be miscible with the supercritical fluid.In some embodiments, the modifier is a liquid at ambient conditions. Byway of example and without limitation, a modifier can be selected fromthe group consisting of ethanol, methanol, propanol, acetone, ethylacetate, methylene chloride, etc.

The extract is a mixture of pharmacologically active compounds, such asoleandrin or other cardiac glycosides, oleaside, and other plantmaterials. Oleandrin extract from a supercritical fluid process containsby weight a theoretical range of 0.9% to 2.5% oleandrin. SCF extractscomprising varying amount of oleandrin have been obtained. In oneembodiment, the SCF extract comprises about 2% by wt. of oleandrin.

The SCF extract can comprise a mixture of various components. Some ofthose components include oleandrin, oleaside A, oleandrigenin,neritaloside, odorside (Wang X, Plomley J B, Newman R A and Cisneros A.LC/MS/MS analyses of an oleander extract for cancer treatment,Analytical Chem. 72: 3547-3552, 2000), and other unidentifiedcomponents. The SCF extractable unidentified components of the SCFextract can include at least one other cardiac glycosidepharmacologically active component and/or at least one other non-cardiacglycoside pharmacologically active component that contributes to theefficacy of the oleandrin in the SCF extract. That is, at least oneother SCF extractable component functions additively or synergisticallywith the oleandrin to provide the observed efficacy.

It has been determined that extract comprising oleandrin or oleandrinand one or more other pharmacologically active components (cardiacglycoside and/or non-cardiac glycoside) can provide neuroprotection.Accordingly, the SCF extract of the invention comprises (or consistsessentially of) oleandrin and one or more other biologically activecomponents, which themselves can be a cardiac glycoside or a non-cardiacglycoside, that provide neuroprotection.

Further evidence of the existence of one or more biologically activecomponents, other than oleandrin, in the SCF extract was obtained bycomparing the concentration-response curves for an aqueous solutioncontaining pure oleandrin versus one containing the SCF extract. FIG. 1Ddepicts the results of a concentration-response assay for an aqueoussolution containing pure oleandrin in a neuroprotectionbrain-slice-based “stroke” assay as described in Example 11. Theconcentration of oleandrin in the aqueous solution was varied from0.0069 to 230 μg/ml.

FIG. 1E depicts results of a concentration-response assay for anoleandrin-containing SCF extract in a neuroprotection brain-slice-based“stroke” assay as described herein. The data demonstrate that theextract is more efficacious that pure oleandrin meaning the extractcontains one or more pharmacologically active agents that provideneuroprotection.

It is possible that the extracts also differ in their relativeperformance as determined by efficacy in the assays included herein.Even so, if a cardiac glycoside is present in a sufficiently high amountor concentration in the extract to be able to prepare a therapeuticallyrelevant dose, then the extract is considered part of the invention.

The cardiac glycoside can be formulated in any suitable pharmaceuticallyacceptable dosage form. Parenteral, otic, ophthalmic, nasal, inhalable,buccal, sublingual, enteral, topical, oral, peroral, and injectabledosage forms are particularly useful. Particular dosage forms include asolid or liquid dosage forms. Exemplary suitable dosage forms includetablet, capsule, pill, caplet, troche, sache, solution, suspension,dispersion, vial, bag, bottle, injectable liquid, i.v. (intravenous),i.m. (intramuscular) or i.p. (intraperitoneal) administrable liquid andother such dosage forms known to the artisan of ordinary skill in thepharmaceutical sciences.

The amount of cardiac glycoside incorporated in a dose of the inventionwill be at least one or more dosage forms and can be selected accordingto known principles of pharmacy. An effective amount or therapeuticallyrelevant (effective) amount of therapeutic compound is specificallycontemplated. By the term “effective amount”, it is understood that,with respect to, for example, pharmaceuticals, a pharmaceuticallyeffective amount is contemplated. A pharmaceutically effective amount isthe amount or quantity of active ingredient which is enough for therequired or desired therapeutic response, or in other words, the amount,which is sufficient to elicit an appreciable biological response when,administered to a patient. The appreciable biological response may occuras a result of administration of single or multiple doses of an activesubstance. A dose may comprise one or more dosage forms. It will beunderstood that the specific dose level for any patient will depend upona variety of factors including the indication being treated, severity ofthe indication, patient health, age, gender, weight, diet,pharmacological response, the specific dosage form employed, and othersuch factors.

The desired dose for oral administration is up to 5 dosage formsalthough as few as one and as many as ten dosage forms may beadministered. Exemplary dosage forms contain 0.6 mg of the SCF extractper dosage form, for a total 0.6 to 60 mg (1 to 10 dose levels) perdose.

The cardiac glycoside can be present in a dosage form in an amountsufficient to provide a subject with an initial dose of oleandrin of 12to 1200 ug, or more or less. A dosage form can comprise 0.01 to 100 mgof oleandrin, oleandrin extract or extract of Nerium oleander containingoleandrin.

For use in treatment of mammals, the cardiac glycoside can be includedin a dosage form. Some embodiments of the dosage form are not entericcoated and release their charge of cardiac glycoside within a period of0.5 to 1 hours or less. Some embodiments of the dosage form are entericcoated and release their charge of cardiac downstream of the stomach,such as from the jejunum, ileum, small intestine, and/or large intestine(colon). Enterically coated dosage forms will release cardiac glycosidesinto the systemic circulation within 1-10 hr after oral administration.

Based on preliminary animal dosing data it is anticipated that 50 to 75%of an administered dose of oleander extract will be orally bioavailabletherefore providing 0.25 to 0.4 mg, 0.1 to 50 mg, 0.1 to 40 mg, 0.2 to40 mg, 0.2 to 30 mg, 0.2 to 20 mg, 0.2 to 10 mg, 0.2 to 5 mg, 0.2 to 2.5mg, 0.2 to 2 mg, 0.2 to 1.5 mg, 0.2 to 1 mg, 0.2 to 0.8 mg, 0.2 to 0.7,or 0.25 to 0.5 mg of oleandrin per dosage form. Given an average bloodvolume in adult humans of 5 liters, the anticipated oleandrin plasmaconcentration will be in the range of 0.05 to 2 ng/ml, 0.005 to 10ng/mL, 0.005 to 8 ng/mL, 0.01 to 7 ng/mL, 0.02 to 7 ng/mL, 0.03 to 6ng/mL, 0.04 to 5 ng/mL, or 0.05 to 2.5 ng/mL. The recommended daily doseof oleandrin, present in the SCF extract, is generally about 0.25 toabout 50 mg twice daily or about 0.9 to 5 mg twice daily or about every12 hours. The dose can be about 0.5 to about 100 mg/day, about 1 toabout 80 mg/day, about 1.5 to about 60 mg/day, about 1.8 to about 60mg/day, about 1.8 to about 40 mg/day. The maximum tolerated dose can beabout 100 mg/day, about 80 mg/day, about 60 mg/day, about 40 mg/day,about 38.4 mg/day or about 30 mg/day of oleander extract containingoleandrin and the minimum effective dose can be about 0.5 mg/day, about1 mg/day, about 1.5 mg/day, about 1.8 mg/day, about 2 mg/day, or about 5mg/day.

It should be noted that a compound herein might possess one or morefunctions in the formulation of the invention. For example, a compoundmight serve as both a surfactant and a water miscible solvent or as botha surfactant and a water immiscible solvent.

A liquid composition can comprise one or more pharmaceuticallyacceptable liquid carriers. The liquid carrier can be an aqueous,non-aqueous, polar, non-polar, and/or organic carrier. Liquid carriersinclude, by way of example and without limitation, a water misciblesolvent, water immiscible solvent, water, buffer and mixtures thereof.

As used herein, the terms “water soluble solvent” or “water misciblesolvent”, which terms are used interchangeably, refer to an organicliquid which does not form a biphasic mixture with water or issufficiently soluble in water to provide an aqueous solvent mixturecontaining at least five percent of solvent without separation of liquidphases. The solvent is suitable for administration to humans or animals.Exemplary water soluble solvents include, by way of example and withoutlimitation, PEG (poly(ethylene glycol)), PEG 400 (poly(ethylene glycolhaving an approximate molecular weight of about 400), ethanol, acetone,alkanol, alcohol, ether, propylene glycol, glycerin, triacetin,poly(propylene glycol), PVP (poly(vinyl pyrrolidone)),dimethylsulfoxide, N,N-dimethylformamide, formamide,N,N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol,soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone).

As used herein, the terms “water insoluble solvent” or “water immisciblesolvent”, which terms are used interchangeably, refer to an organicliquid which forms a biphasic mixture with water or provides a phaseseparation when the concentration of solvent in water exceeds fivepercent. The solvent is suitable for administration to humans oranimals. Exemplary water insoluble solvents include, by way of exampleand without limitation, medium/long chain triglycerides, oil, castoroil, corn oil, vitamin E, vitamin E derivative, oleic acid, fatty acid,olive oil, softisan 645 (Diglyceryl Caprylate/Caprate/Stearate/Hydroxystearate adipate), miglyol, captex (Captex 350: GlycerylTricaprylate/Caprate/Laurate triglyceride; Captex 355: GlycerylTricaprylate/Caprate triglyceride; Captex 355 EP/NF: GlycerylTricaprylate/Caprate medium chain triglyceride).

Suitable solvents are listed in the “International Conference onHarmonisation of Technical Requirements for Registration ofPharmaceuticals for Human Use (ICH) guidance for industry Q3CImpurities: Residual Solvents” (1997), which makes recommendations as towhat amounts of residual solvents are considered safe inpharmaceuticals. Exemplary solvents are listed as class 2 or class 3solvents. Class 3 solvents include, for example, acetic acid, acetone,anisole, 1-butanol, 2-butanol, butyl acetate, tert-butlymethyl ether,cumene, ethanol, ethyl ether, ethyl acetate, ethyl formate, formic acid,heptane, isobutyl acetate, isopropyl acetate, methyl acetate,methyl-1-butanol, methylethyl ketone, methylisobutyl ketone,2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, orpropyl acetate.

Other materials that can be used as water immiscible solvents in theinvention include: Captex 100: Propylene Glycol Dicaprate; Captex 200:Propylene Glycol Dicaprylate/Dicaprate; Captex 200 P: Propylene GlycolDicaprylate/Dicaprate; Propylene Glycol Dicaprylocaprate; Captex 300:Glyceryl Tricaprylate/Caprate; Captex 300 EP/NF: GlycerylTricaprylate/Caprate Medium Chain Triglycerides; Captex 350: GlycerylTricaprylate/Caprate/Laurate; Captex 355: Glyceryl Tricaprylate/Caprate;Captex 355 EP/NF: Glyceryl Tricaprylate/Caprate Medium ChainTriglycerides; Captex 500: Triacetin; Captex 500 P: Triacetin(Pharmaceutical Grade); Captex 800: Propylene Glycol Di(2-Ethylhexanoate); Captex 810 D: GlycerylTricaprylate/Caprate/Linoleate; Captex 1000: Glyceryl Tricaprate; CaptexCA: Medium Chain Triglycerides; Captex MCT-170: Medium ChainTriglycerides; Capmul GMO: Glyceryl Monooleate; Capmul GMO-50 EP/NF:Glyceryl Monooleate; Capmul MCM: Medium Chain Mono- & Diglycerides;Capmul MCM C8: Glyceryl Monocaprylate; Capmul MCM C10: GlycerylMonocaprate; Capmul PG-8: Propylene Glycol Monocaprylate; Capmul PG-12:Propylene Glycol Monolaurate; Caprol 100100: Decaglycerol Decaoleate;Caprol 3GO: Triglycerol Monooleate; Caprol ET: Polyglycerol Ester ofMixed Fatty Acids; Caprol MPGO: Hexaglycerol Dioleate; Caprol PGE 860:Decaglycerol Mono-, Dioleate.

As used herein, a “surfactant” refers to a compound that comprises polaror charged hydrophilic moieties as well as non-polar hydrophobic(lipophilic) moieties; i.e., a surfactant is amphiphilic. The termsurfactant may refer to one or a mixture of compounds. A surfactant canbe a solubilizing agent, an emulsifying agent or a dispersing agent. Asurfactant can be hydrophilic or hydrophobic.

The hydrophilic surfactant can be any hydrophilic surfactant suitablefor use in pharmaceutical compositions. Such surfactants can be anionic,cationic, zwitterionic or non-ionic, although non-ionic hydrophilicsurfactants are presently preferred. As discussed above, these non-ionichydrophilic surfactants will generally have HLB values greater thanabout 10. Mixtures of hydrophilic surfactants are also within the scopeof the invention.

Similarly, the hydrophobic surfactant can be any hydrophobic surfactantsuitable for use in pharmaceutical compositions. In general, suitablehydrophobic surfactants will have an HLB value less than about 10.Mixtures of hydrophobic surfactants are also within the scope of theinvention.

Examples of additional suitable solubilizer include: alcohols andpolyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethyleneglycol, propylene glycol, butanediols and isomers thereof, glycerol,pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide,polyethylene glycol, polypropylene glycol, polyvinylalcohol,hydroxypropyl methylcellulose and other cellulose derivatives,cyclodextrins and cyclodextrin derivatives; ethers of polyethyleneglycols having an average molecular weight of about 200 to about 6000,such as tetrahydrofurfuryl alcohol PEG ether (glycofurol, availablecommercially from BASF under the trade name Tetraglycol) or methoxy PEG(Union Carbide); amides, such as 2-pyrrolidone, 2-piperidone,caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone,N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide, andpolyvinypyrrolidone; esters, such as ethyl propionate, tributylcitrate,acetyl triethylcitrate, acetyl tributyl citrate, triethylcitrate, ethyloleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycolmonoacetate, propylene glycol diacetate, caprolactone and isomersthereof, valerolactone and isomers thereof, butyrolactone and isomersthereof; and other solubilizers known in the art, such as dimethylacetamide, dimethyl isosorbide (Arlasolve DMI (ICI)),N-methylpyrrolidones (Pharmasolve (ISP)), monooctanoin, diethyleneglycol nonoethyl ether (available from Gattefosse under the trade nameTranscutol), and water. Mixtures of solubilizers are also within thescope of the invention.

Except as indicated, compounds mentioned herein are readily availablefrom standard commercial sources.

The clear liquid composition is visually clear to the unaided eye, as itwill contain less than 5%, less than 3% or less than 1% by wt. ofsuspended solids based upon the total weight of the composition.

Although not necessary, a composition or kit of the present inventionmay include a chelating agent, preservative, antioxidant, adsorbents,acidifying agent, alkalizing agent, antifoaming agent, buffering agent,colorant, electrolyte, salt, stabilizer, tonicity modifier, diluent,other pharmaceutical excipient, or a combination thereof.

As used herein, the term “antioxidant” is intended to mean an agent thatinhibits oxidation and is thus used to prevent the deterioration ofpreparations by the oxidative process. Such compounds include, by way ofexample and without limitation, ascorbic acid, ascorbic palmitate,Vitamin E, Vitamin E derivative, butylated hydroxyanisole, butylatedhydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate,sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate,sodium metalbisulfite and other such materials known to those ofordinary skill in the art.

As used herein, the term chelating agent is intended to mean a compoundthat chelates metal ions in solution. Exemplary chelating agents includeEDTA (tetrasodium ethylenediaminetetraacetate), DTPA (pentasodiumdiethylenetriamine-pentaacetate), HEDTA (trisodium salt ofN-(hydroxyethyl)-ethylene-diaminetriacetic acid), NTA (trisodiumnitrilotriacetate), disodium ethanoldiglycine (Na₂EDG), sodiumdiethanolglycine (DEGNa), citric acid, and other compounds known tothose of ordinary skill in the art.

As used herein, the term “adsorbent” is intended to mean an agentcapable of holding other molecules onto its surface by physical orchemical (chemisorption) means. Such compounds include, by way ofexample and without limitation, powdered and activated charcoal andother materials known to one of ordinary skill in the art.

As used herein, the term “alkalizing agent” is intended to mean acompound used to provide an alkaline medium. Such compounds include, byway of example and without limitation, ammonia solution, ammoniumcarbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodiumborate, sodium carbonate, sodium bicarbonate, sodium hydroxide,triethanolamine, and trolamine and others known to those of ordinaryskill in the art.

As used herein, the term “acidifying agent” is intended to mean acompound used to provide an acidic medium. Such compounds include, byway of example and without limitation, acetic acid, amino acid, citricacid, fumaric acid and other alpha-hydroxy acids, hydrochloric acid,ascorbic acid, and nitric acid and others known to those of ordinaryskill in the art.

As used herein, the term “antifoaming agent” is intended to mean acompound or compounds that prevents or reduces the amount of foamingthat forms on the surface of the fill composition. Suitable antifoamingagents include by way of example and without limitation, dimethicone,SIMETHICONE, octoxynol and others known to those of ordinary skill inthe art.

As used herein, the term “buffering agent” is intended to mean acompound used to resist a change in pH upon dilution or addition of acidor alkali. Such compounds include, by way of example and withoutlimitation, potassium metaphosphate, potassium phosphate, monobasicsodium acetate and sodium citrate anhydrous and dehydrate and other suchmaterials known to those of ordinary skill in the art.

As used herein, the term “diluent” or “filler” is intended to mean inertsubstances used as fillers to create the desired bulk, flow properties,and compression characteristics in the preparation of tablets andcapsules. Such compounds include, by way of example and withoutlimitation, dibasic calcium phosphate, kaolin, lactose, sucrose,mannitol, microcrystalline cellulose, powdered cellulose, precipitatedcalcium carbonate, sorbitol, and starch and other materials known to oneof ordinary skill in the art.

As used herein, the term “preservative” is intended to mean a compoundused to prevent the growth of microorganisms. Such compounds include, byway of example and without limitation, benzalkonium chloride,benzethonium chloride, benzoic acid, benzyl alcohol, cetylpyridiniumchloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuricnitrate, phenylmercuric acetate, thimerosal, metacresol, myristylgammapicolinium chloride, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, sorbic acid, thymol, and methyl, ethyl,propyl, or butyl parabens and others known to those of ordinary skill inthe art.

As used herein, the term “colorant” is intended to mean a compound usedto impart color to pharmaceutical preparations. Such compounds include,by way of example and without limitation, FD&C Red No. 3, FD&C Red No.20, FD&C Yellow No. 6, FD&C Blue No. 2, FD&C Green No. 5, FD&C OrangeNo. 5, FD&C Red No. 8, caramel, and iron oxide (black, red, yellow),other FD&C dyes and natural coloring agents such as grape skin extract,beet red powder, beta-carotene, annato, carmine, turmeric, paprika,combinations thereof and other such materials known to those of ordinaryskill in the art.

As used herein, the term “stabilizer” is intended to mean a compoundused to stabilize an active agent against physical, chemical, orbiochemical processes that would otherwise reduce the therapeuticactivity of the agent. Suitable stabilizers include, by way of exampleand without limitation, albumin, sialic acid, creatinine, glycine andother amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide,sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethyleneglycols, sodium caprylate and sodium saccharin and others known to thoseof ordinary skill in the art.

As used herein, the term “tonicity modifier” is intended to mean acompound or compounds that can be used to adjust the tonicity of theliquid formulation. Suitable tonicity modifiers include glycerin,lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol,trehalose and others known to those or ordinary skill in the art.

The composition of the invention can also include oils such as fixedoils, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil;fatty acids such as oleic acid, stearic acid and isostearic acid; andfatty acid esters such as ethyl oleate, isopropyl myristate, fatty acidglycerides and acetylated fatty acid glycerides. The composition canalso include alcohol such as ethanol, isopropanol, hexadecyl alcohol,glycerol and propylene glycol; glycerol ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol; ethers such as poly(ethyleneglycol) 450; petroleum hydrocarbons such as mineral oil and petrolatum;water; a pharmaceutically suitable surfactant, suspending agent oremulsifying agent; or mixtures thereof.

It should be understood that the compounds used in the art ofpharmaceutical formulation generally serve a variety of functions orpurposes. Thus, if a compound named herein is mentioned only once or isused to define more than one term herein, its purpose or function shouldnot be construed as being limited solely to that named purpose(s) orfunction(s).

One or more of the components of the formulation can be present in itsfree base or pharmaceutically or analytically acceptable salt form. Asused herein, “pharmaceutically or analytically acceptable salt” refersto a compound that has been modified by reacting it with an acid asneeded to form an ionically bound pair. Examples of acceptable saltsinclude conventional non-toxic salts formed, for example, from non-toxicinorganic or organic acids. Suitable non-toxic salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfonic, sulfamic, phosphoric, nitric and others known tothose of ordinary skill in the art. The salts prepared from organicacids such as amino acids, acetic, propionic, succinic, glycolic,stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and others known to those of ordinaryskill in the art. Lists of other suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th). ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, the relevant disclosure of which is herebyincorporated by reference.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith tissues of human beings and animals and without excessive toxicity,irritation, allergic response, or any other problem or complication,commensurate with a reasonable benefit/risk ratio.

A dosage form can be made by any conventional means known in thepharmaceutical industry. A liquid dosage form can be prepared byproviding at least one liquid carrier and oleandrin oroleandrin-containing extract in a container. One or more otherexcipients can be included in the liquid dosage form. A solid dosageform can be prepared by providing at least one solid carrier andoleandrin or oleandrin-containing extract. One or more other excipientscan be included in the solid dosage form.

A dosage form can be packaged using conventional packaging equipment andmaterials. It can be included in a pack, bottle, via, bag, syringe,envelope, packet, blister pack, box, ampoule, or other such container.

The invention includes a method for improving the clinical status of astatistically significant number of subjects of in a population ofsubjects having a neurological condition, the method comprising:administering to the population of subjects a cardiac glycoside orcardiac glycoside-containing composition; and determining the clinicalstatus of the subjects. In some embodiments, the statisticallysignificant number is at least 5%, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80% or at least 90% of the population. In some embodiments, thecomposition comprises oleandrin or an extract comprising oleandrin. Theextract optionally comprises one or more other pharmacologically activecompounds that cooperate with oleandrin to improve the clinical statusof the subjects.

As used herein a “derivative” is: a) a chemical substance that isrelated structurally to a first chemical substance and theoreticallyderivable from it; b) a compound that is formed from a similar firstcompound or a compound that can be imagined to arise from another firstcompound, if one atom of the first compound is replaced with anotheratom or group of atoms; c) a compound derived or obtained from a parentcompound and containing essential elements of the parent compound; or d)a chemical compound that may be produced from first compound of similarstructure in one or more steps.

In view of the above description and the examples below, one of ordinaryskill in the art will be able to practice the invention as claimedwithout undue experimentation. The foregoing will be better understoodwith reference to the following examples that detail certain proceduresfor the preparation of embodiments of the present invention. Allreferences made to these examples are for the purposes of illustration.The following examples should not be considered exhaustive, but merelyillustrative of only a few of the many embodiments contemplated by thepresent invention.

Oleandrin can be purchased from Sigma Chemical Co. (St. Louis, Mo.).

Example 1 Supercritical Fluid Extraction of Powdered Oleander LeavesMethod A. With Carbon Dioxide.

Powdered oleander leaves were prepared by harvesting, washing, anddrying oleander leaf material, then passing the oleander leaf materialthrough a comminuting and dehydrating apparatus such as those describedin U.S. Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. Theweight of the starting material used was 3.94 kg.

The starting material was combined with pure CO₂ at a pressure of 300bar (30 MPa, 4351 psi) and a temperature of 50° C. (122° F.) in anextractor device. A total of 197 kg of CO₂ was used, to give a solventto raw material ratio of 50:1. The mixture of CO₂ and raw material wasthen passed through a separator device, which changed the pressure andtemperature of the mixture and separated the extract from the carbondioxide.

The extract (65 g) was obtained as a brownish, sticky, viscous materialhaving a nice fragrance. The color was likely caused by chlorophyll. Foran exact yield determination, the tubes and separator were rinsed outwith acetone and the acetone was evaporated to give an addition 9 g ofextract. The total extract amount was 74 g. Based on the weight of thestarting material, the yield of the extract was 1.88%. The content ofoleandrin in the extract was calculated using high pressure liquidchromatography and mass spectrometry to be 560.1 mg, or a yield of0.76%.

Method B. With Mixture of Carbon Dioxide and Ethanol

Powdered oleander leaves were prepared by harvesting, washing, anddrying oleander leaf material, then passing the oleander leaf materialthrough a comminuting and dehydrating apparatus such as those describedin U.S. Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. Theweight of the starting material used was 3.85 kg.

The starting material was combined with pure CO₂ and 5% ethanol as amodifier at a pressure of 280 bar (28 MPa, 4061 psi) and a temperatureof 50° C. (122° F.) in an extractor device. A total of 160 kg of CO₂ and8 kg ethanol was used, to give a solvent to raw material ratio of 43.6to 1. The mixture of CO₂, ethanol, and raw material was then passedthrough a separator device, which changed the pressure and temperatureof the mixture and separated the extract from the carbon dioxide.

The extract (207 g) was obtained after the removal of ethanol as a darkgreen, sticky, viscous mass obviously containing some chlorophyll. Basedon the weight of the starting material, the yield of the extract was5.38%. The content of oleandrin in the extract was calculated using highpressure liquid chromatography and mass spectrometry to be 1.89 g, or ayield of 2.1%.

Example 2 Hot-Water Extraction of Powdered Oleander Leaves

Hot water extraction is typically used to extract oleandrin and otheractive components from oleander leaves. Examples of hot water extractionprocesses can be found in U.S. Pat. Nos. 5,135,745 and 5,869,060.

A hot water extraction was carried out using 5 g of powdered oleanderleaves. Ten volumes of boiling water (by weight of the oleander startingmaterial) were added to the powdered oleander leaves and the mixture wasstirred constantly for 6 hours. The mixture was then filtered and theleaf residue was collected and extracted again under the sameconditions. The filtrates were combined and lyophilized. The appearanceof the extract was brown. The dried extract material weighed about 1.44g. 34.21 mg of the extract material was dissolved in water and subjectedto oleandrin content analysis using high pressure liquid chromatographyand mass spectrometry. The amount of oleandrin was determined to be 3.68mg. The oleandrin yield, based on the amount of extract, was calculatedto be 0.26%. The table below shows a comparison between the oleandrinyields for the two supercritical carbon dioxide extractions of Example 1and the hot water extraction.

Comparison of Yields

Oleandrin yield based Extraction Medium on total extract weightSupercritical Carbon Dioxide: Example 1, 0.76% Method A SupercriticalCarbon Dioxide: Example 1, 2.1% Method B Hot Water Extraction: Example 20.26%

Example 3 Treatment of Neurological Condition Including but not Limitedto Alzheimer's Disease Method A. Cardiac Glycoside Therapy

A subject presenting with Alzheimer's disease is prescribed cardiacglycoside, and therapeutically relevant doses are administered to thesubject according to a prescribed dosing regimen for a period of time.The subject's level of therapeutic response is determined periodically.If the level of therapeutic response is too low at one dose, then thedose is escalated according to a predetermined dose escalation scheduleuntil the desired level of therapeutic response in the subject isachieved. Treatment of the subject with cardiac glycoside is continuedas needed and the dose or dosing regimen can be adjusted as needed untilthe patient reaches the desired clinical endpoint.

Method B. Combination Therapy: Cardiac Glycoside with Another Agent

Method A, above, is followed except that the subject is prescribed andadministered one or more other therapeutic agents for the treatment ofAlzheimer's disease, or symptoms thereof. Then one or more othertherapeutic agents can be administered before, after or with the cardiacglycoside. Dose escalation (or de-escalation) of the one or more othertherapeutic agents can also be done. Suitable one or more othertherapeutic agents include Namenda™ (memantine HCl), Aricept™(donepezil), Razadyne™ (galantamine), Exelon™ (rivastigmine), andCognex™ (tacrine).

Example 4 Treatment of Neurological Condition Including but not Limitedto Huntington's Disease Method A. Cardiac Glycoside Therapy

A subject presenting with Huntington's disease is prescribed cardiacglycoside, and therapeutically relevant doses are administered to thesubject according to a prescribed dosing regimen for a period of time.The subject's level of therapeutic response is determined periodically.If the level of therapeutic response is too low at one dose, then thedose is escalated according to a predetermined dose escalation scheduleuntil the desired level of therapeutic response in the subject isachieved. Treatment of the subject with cardiac glycoside is continuedas needed and the dose or dosing regimen can be adjusted as needed untilthe patient reaches the desired clinical endpoint. The dosesadministered can be similar to those of Example 3 or as otherwisedescribed herein.

Method B. Combination Therapy: Cardiac Glycoside with Another Agent

Method A, above, is followed except that the subject is prescribed andadministered one or more other therapeutic agents for the treatment ofHuntington's disease, or symptoms thereof. The one or more othertherapeutic agents can be administered before, after or with the cardiacglycoside. Dose escalation (or de-escalation) of the one or more othertherapeutic agents can also be done. Suitable one or more othertherapeutic agents include Vitamin E, Baclofen (a derivative of CoQ10),Lamotrigine (an anticonvulsant), remacemide (an anesthetic which is alow affinity NMDA antagonist), and riluzole (Na channel blocker).

Example 5 Treatment of Neurological Condition Including but not Limitedto Ischemic Stroke Method A. Cardiac Glycoside Therapy

A subject presenting with ischemic stroke is prescribed cardiacglycoside, and therapeutically relevant doses are administered to thesubject according to a prescribed dosing regimen for a period of time.The subject's level of therapeutic response is determined periodically.If the level of therapeutic response is too low at one dose, then thedose is escalated according to a predetermined dose escalation scheduleuntil the desired level of therapeutic response in the subject isachieved. Treatment of the subject with cardiac glycoside is continuedas needed and the dose or dosing regimen can be adjusted as needed untilthe patient reaches the desired clinical endpoint. The dosesadministered can be similar to those in Example 3 or as otherwisedescribed herein.

Method B. Combination Therapy: Cardiac Glycoside with Another Agent

Method A, above, is followed except that the subject is prescribed andadministered one or more other therapeutic agents for the treatment ofischemic stroke, or symptoms thereof. The one or more other therapeuticagents can be administered before, after or with the cardiac glycoside.Dose escalation (or de-escalation) of the one or more other therapeuticagents can also be done.

Example 6 HPLC Analysis of Solutions Containing Oleandrin

Samples (oleandrin standard, SCF extract and hot-water extract) wereanalyzed on HPLC (Waters) using the following conditions: Symmetry C18column (5.0 μm, 150×4.6 mm I.D.; Waters); Mobile phase of MeOH:water=54:46 (v/v) and flow rate at 1.0 ml/min Detection wavelength was set at 217nm. The samples were prepared by dissolving the compound or extract in afixed amount of HPLC solvent to achieve an approximate targetconcentration of oleandrin.

Example 7 Determination of α3 and α1 Expression in Normal NeuronalTissue

The procedures set forth in PCT International Application No.PCT/US08/82641, filed Nov. 6, 2008 in the name of Phoenix Biotechnology,Inc., the entire disclosure of which is hereby incorporated byreference, can be followed.

Example 8 Evaluation of a Cardiac Glycoside in an In Vitro Assay forStroke and Non-Stroke Method A. Stroke: Preparation of Cortical BrainSlices and OGD.

Neocortical brain slices were prepared from PND 7 Sprague-Dawley ratpups. The cerebral cortex was dissected, cut into 400-_m-thick slicesand transferred into a container containing cold artificialcerebrospinal fluid with 1 uM MK-801 before plating; MK-801 was notincluded in any subsequent procedures. To mimic ischemic injury usingtransient oxygen-glucose deprivation (OGD), slices from one hemisphereof each brain were exposed to glucose-free, N₂-bubbled artificialcerebrospinal fluid for 7.5 min in a low O₂ (0.5%) environment. The OGDslices were then plated side-by-side with control slices from thecontralateral hemisphere on nitrocellulose or Millicell (Millipore)permeable membranes, which were prepared identically except for no OGD.Thirty minutes after plating, the brain slice pairs were transfected,transferred to 24-well plates, and incubated at 37° C. under 5% CO₂ inhumidified chambers. In each experiment, 5-6 minutes of oxygen-glucosedeprivation (OGD) was used to induce >50% loss of healthy corticalneurons by 24 hrs (compare first two bars in FIG. 1A). A setconcentration (3 μM) of neriifolin was used as the internal positivecontrol. For oleandrin, all three concentrations from 0.3 to 3 μMappeared to provide neuroprotection in the first two experiments (FIGS.1A and 1B), so the oleandrin concentrations tested were lowered in thethird run (FIG. 1C) and suggested that the threshold concentration forneuroprotection lies between 0.1 and 0.3 μM.

Method B. Non-Stroke: Brain Slice Assay.

Oleandrin and PBI-05204 were tested on “nonstroked” brain slices; thatis, ones that were sliced and transfected with YFP but not subjected toadditional trauma via OGD. See experimental procedure outlined above. Wehave observed that a number of neuroprotective compounds, includingneriifolin, can provide modest levels of neuroprotection to such brainslices, presumably by protecting against the trauma caused by theprocess of slicing and culturing itself. As can be seen in FIGS. 2A-2Coleandrin appeared to be able to provide neuroprotection to such“non-OGD” brain slices to similar levels as neriifolin signifying thatcardiac glycosides mediate neuroprotection even in the absence of oxygenor glucose deprivation.

Example 9 Evaluation of a Cardiac Glycoside in an In Vitro APP Assay forAlzheimer's Disease

In the rat brain slice model for APP/Abeta-induced degeneration ofcortical pyramidal neurons biolistic transfection is used not only tointroduce vital markers such as YFP, but also to introduce disease geneconstructs into the same neuronal populations in the brain slices. Thus,the APP/Aβ brain slice model co-transfects YFP with APP isoforms,leading to the progressive degeneration of cortical pyramidal neuronsover the course of 3-4 days after brain slice preparation andtransfection. As can be seen in the three runs in FIGS. 3A-3C, botholeandrin and PBI-05204 appeared able to provide dose-dependentneuroprotection to APP-transfected brain slices, rescuing to levelsnearly to those that can be provided by BACE inhibitor drugs.

Example 10 Evaluation of a Cardiac Glycoside in an In VitroCorticostriatal Co-Culture Assay for Huntington's Disease

In this assay, instead of using intact brain slices, mutant htt isintroduced via electroporation into high-density, mixed co-cultures ofcortical neurons, striatal neurons, and glia arrayed in 96-well plates.The goal of this assay platform is to combine the biological/clinicalrelevance of a complex primary culture system that recapitulates keyaspects of the interconnectivity of disease-relevant neuronalpopulations in vivo, with the ability to conduct large-scale fullyautomated screening campaigns. In this assay, over the course of 1-2weeks in vitro, transfected mutant htt constructs induce the progressivedegeneration of both striatal and cortical neurons that are subsequentlyquantified using automated image acquisition and object detectionalgorithms on the Cellomics Arrayscan VTI platform. Each data point wasdrawn from 6 wells with 16 images in each well automatically captured,processed, and analyzed on the Cellomics Arrayscan using protocolsdeveloped during a large-scale screening campaign being conducted inassociation with the Cure Huntington's Disease Initiative. In a fullrun, some 25,000 images are collected and analyzed in each cycle, 4cycles per week.

Cortico-Striatal Co-Culture Assay Platform.

Pure glial cultures are prepared in advance of neuronal plating toestablish 96-well plates with confluent glial beds. Cortical andstriatal tissue are then dissociated separately and “nucleofected” withappropriate DNA constructs and are distinguishable later by theexpression of different fluorescent proteins such as YFP, CFP, andmCherry. These separately transfected cortical and striatal neurons arethen mixed thoroughly and plated into the 96-well plates containing thepreviously plated glial monolayers.

Both oleandrin and PBI-05204 (the supercritical CO₂ extract of Neriumoleander) were tested in this cortico-striatal co-culture platform andpreliminarily these compounds appear to be the strongest hits we haveobserved to date out of >400 late-stage drug molecules that have beenevaluated in this assay system. For comparison, a dose-response graphfor KW6002 (an adenosine 2a receptor antagonist), the compound that weroutinely include as the positive control for this co-culture assay isincluded (see FIG. 4E). Efficacy of oleandrin is on par with KW6002,while its potency appears to be some 100-fold greater.

Example 11 Evaluation of a Cardiac Glycoside and an Extract of theInvention in an In Vitro Assay for Stroke and Non-Stroke Method A.Stroke: Preparation of Cortical Brain Slices and OGD.

Neocortical brain slices were prepared from PND 7 Sprague-Dawley ratpups. The cerebral cortex was dissected, cut into 400-μ-thick slices andtransferred into a container containing cold artificial cerebrospinalfluid with 1 uM MK-801 before plating; MK-801 was not included in anysubsequent procedures. To mimic ischemic injury using transientoxygen-glucose deprivation (OGD), slices from one hemisphere of eachbrain were exposed to glucose-free, N₂-bubbled artificial cerebrospinalfluid for 7.5 min in a low O₂ (0.5%) environment. The OGD slices werethen plated side-by-side with control slices from the contralateralhemisphere on nitrocellulose or Millicell (Millipore) permeablemembranes, which were prepared identically except for no OGD. Thirtyminutes after plating, the brain slice pairs were transfected,transferred to 24-well plates, and incubated at 37° C. under 5% CO₂ inhumidified chambers. In each experiment, 5-6 minutes of oxygen-glucosedeprivation (OGD) was used to induce >50% loss of healthy corticalneurons by 24 hrs. A set concentration (3 μM) of neriifolin (a cardiacglycoside) was used as the internal positive control. For oleandrin (acardiac glycoside), all three concentrations from 0.3 to 3 μM appearedto provide neuroprotection in the first two experiments, so theoleandrin concentrations tested were lowered in the third run andsuggested that the threshold concentration for neuroprotection liesbetween 0.1 and 0.3 μM. The unfractionated extract, e.g. of Neriumspecies, or a fraction thereof can also be used as described for theoleandrin.

Method B. Non-Stroke: Brain Slice Assay.

Oleandrin and PBI-05204, an unfractionated SCF extract of Neriumoleander, were tested on “nonstroked” brain slices; that is, ones thatwere sliced and transfected with YFP but not subjected to additionaltrauma via OGD. See experimental procedure outlined above. We haveobserved that a number of neuroprotective compounds, includingneriifolin, can provide modest levels of neuroprotection to such brainslices, presumably by protecting against the trauma caused by theprocess of slicing and culturing itself. The data demonstrate thatoleandrin and the extract appeared to be able to provide neuroprotectionto such “non-OGD” brain slices to similar levels as neriifolinsignifying that cardiac glycosides mediate neuroprotection even in theabsence of oxygen or glucose deprivation.

Example 12 HPLC Analysis of SCF Extract

The purpose of this assay is to identify extract containing cardiacglycoside. A sample from each extract was analyzed as follows. Theextract (1-3 mg) was dissolved in 1-5 ml of aqueous methanol (80%methanol in water). The diluted sample (10-25 μl) was analyzed with anAgilent Zorbax SB-C18 column using 80% methanol in water as the mobilephase, a flow rate of 0.7 mL/min and DAD-UV effluent monitoring at thefollowing wavelengths: 203, 210, 217, 230, 254, 280, 310 and 300 nmPositive identification is confirmed via peak formation on achromatogram when comparing retention times and spectra of extractsamples to reference samples.

Example 13 Time-Delay Brain-Slice Assay for Determination ofNeuroprotection

This assay was conducted according to Example 11 except that thefollowing changes were made. A specified length of time was allowedbetween OGD and introduction of a proposed neuroprotective agent. Theability of PBI-05204 to provide neuroprotection to brain slices iftreatment was delayed relative to the timing of the OGD treatment wasdetermined. Data showed that a 2 hr delay of Nerium oleander extractswas well tolerated, showing similar levels of neuroprotection to thoseattained with application of PBI-05204 immediately following OGDtreatment. Neuroprotective benefit was reduced with 4 to 6 hr of delayof administration of PBI-05204, but at levels of neuroprotection thatwere still significantly and physiologically relevant.

As used herein, the term “about” or “approximately” are taken tomean±10%, ±5%, ±2.5% or ±1% of a specified valued. As used herein, theterm “substantially” is taken to mean “to a large degree” or “at least amajority of” or “more than 50% of”.

The above is a detailed description of particular embodiments of theinvention. It will be appreciated that, although specific embodiments ofthe invention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims. All of the embodiments disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure.

1) A method of treating a neurological condition in a subjectcomprising: administering to a subject in need thereof a compositioncomprising oleandrin in an effective amount to treat said neurologicalcondition, wherein the composition provides improved treatment ascompared to administration of an equivalent dose of neriifolin. 2) Themethod of claim 1 comprising: determining whether or not theneurological condition in the subject is Alzheimer's disease,Huntington's disease, stroke or other neurological condition; indicatingadministration of oleandrin; administering an initial dose of oleandrinto the subject according to a prescribed initial dosing regimen for aperiod of time; periodically determining the adequacy of subject'sclinical response and/or therapeutic response to treatment witholeandrin; and if the subject's clinical response and/or therapeuticresponse is adequate, then continuing treatment with oleandrin as neededuntil the desired clinical endpoint is achieved; or if the subject'sclinical response and/or therapeutic response are inadequate at theinitial dose and initial dosing regimen, then escalating or deescalatingthe dose until the desired clinical response and/or therapeutic responsein the subject is achieved. 3) The method of claim 1 wherein theneurological condition is a neurological disease or disorder having anetiology associated with altered Na,K-ATPase activity with a compositioncomprising oleandrin, the method comprising: determining that thesubject has a neurological disease or disorder having an etiologyassociated with altered Na,K-ATPase α3 isoform to α1 isoform subunitratio or associated with altered Na,K-ATPase activity; and indicatingadministration to the subject a composition comprising oleandrin. 4) Themethod of claim 1 wherein the neurological condition is a neurologicaldisease or disorder having an etiology associated with altered HIF-1aactivity with a composition comprising oleandrin, the method comprising:determining that the subject has a neurological disease or disorderhaving an etiology associated with altered HIF-1α activity; andindicating administration to the subject a composition comprisingoleandrin. 5) A method for improving the clinical status of astatistically significant number of subjects in a population of subjectshaving a neurological condition, the method comprising: administering tothe population of subjects an oleandrin-containing composition; anddetermining the clinical status of the subjects; wherein the compositionprovides improved clinical status as compared to administration of anequivalent dose of neriifolin. 6) A method of reducing the incidence ofoccurrence of a neurological condition in a population of subjects atrisk thereof, the method comprising administering an effective dose ofoleandrin on a recurring basis for an extended period of time to one ormore subjects in a population of subjects at risk of suffering from aneurological condition selected from Alzheimer's disease, Huntington'sdisease, stroke or other neurological condition, thereby reducing theincidence of the neurological condition in the population; wherein thecomposition provides enhanced reduction in the incidence of occurrenceas compared to administration of an equivalent dose of neriifolin. 7) Atime-delayed method of treating stroke in a subject comprising: withinan acceptable delay period after a subject has suffered the stroke,administering an initial dose of oleandrin according to an initialdosing regimen; determining the adequacy of subject's clinical responseand/or therapeutic response to treatment with oleandrin; and if thesubject's clinical response and/or therapeutic response is adequate,then continuing treatment with oleandrin as needed until the desiredclinical endpoint is achieved; or if the subject's clinical responseand/or therapeutic response are inadequate at the initial dose andinitial dosing regimen, then escalating or deescalating the dose untilthe desired clinical response and/or therapeutic response in the subjectis achieved; wherein the composition provides improved treatment ascompared to administration of an equivalent dose of neriifolin. 8) Theinvention according to any one of the above claims, wherein, followingadministration thereof, oleandrin crosses the blood brain barrier and isretained in brain tissue for a period of at least 8 hours. 9) Theinvention according to any one of the above claims wherein theneurological condition is ischemic stroke or stroke-mediated ischemicbrain injury. 10) The invention according to any one of the above claimswherein the subject is prescribed and administered a therapeuticallyeffective dose of composition comprising oleandrin. 11) The inventionaccording to any one of the above claims wherein administration of thecomposition provides a plasma concentration of oleandrin in the subjectin the range of 0.005 to 10 ng/ml. 12) The invention according to anyone of the above claims wherein the composition comprises 0.1 to 50 mgof oleandrin. 13) The invention according to any one of the above claimswherein a subject is administered a dose of 12 to 1200 μg of oleandrin.14) The invention according to any one of the above claims wherein thesubject is administered the composition comprising oleandrin accordingto a prescribed dosing regimen. 15) The invention according to any oneof the above claims wherein the composition further comprises one ormore other therapeutically effective agents. 16) The invention accordingto any one of the above claims wherein the recurring basis is daily,every other day, every second day, every third day, every fourth day,every fifth day, every sixth day, weekly, every other week, every secondweek, every third week, monthly, bimonthly, semi-monthly, every othermonth every second month, quarterly, every other quarter, trimesterly,seasonally, semi-annually and/or annually. 17) The invention accordingto any one of the above claims wherein the extended period is one ormore weeks, one or more months, one or more quarters and/or one or moreyears. 18) The invention according to any one of the above claimswherein the effective dose is administered one or more times in a day.19) The invention according to any one of the above claims wherein themethod further comprises identifying a population of subjects at risk ofsuffering from a neurological condition selected from Alzheimer'sdisease, Huntington's disease, stroke or other neurological condition.20) The invention according to any one of the above claims wherein thepopulation of subjects at risk is characterized by advancing age of thesubject, familial history of the neurological condition, geneticpredisposition to occurrence of neurological condition, the presence andexpression of ApoE4 gene in the subject, female gender, cardiovasculardisease (e.g. high blood pressure and high cholesterol levels), diabetes(especially Type 2 or adult onset forms of this disease), Down'sSyndrome, head injury, low levels of formal education, smoking,excessive alcohol consumption and/or drug abuse. 21) The inventionaccording to any one of the above claims wherein the delay period is 10hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 3hours or less, 2 hours or less, 1 hour or less, 45 minutes or less, 30minutes or less, 20 minutes or less or 10 min or less. 22) The inventionaccording to any one of the above claims wherein determining theadequacy of a subject's clinical and/or therapeutic response is done byassessments of any weakness of the face, arm and/or leg on one side ofthe body, numbness in the face, arm, and/or leg on one side of the body,inability to understand spoken language, inability to speak or speakclearly, inability to write, vertigo and/or gait imbalance, doublevision and an unusually severe headache. 23) The invention according toany one of the above claims wherein the neurodegenerative disease isselected from the group consisting of Huntington's disease, Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, bovinespongiform encephalopathy, multiple sclerosis, diabetic neuropathy,autism and juvenile neuronal ceroid lipofuscinosis. 24) The inventionaccording to any one of the above claims wherein the oleandrin ispresent in a pharmaceutical formulation or composition. 25) Theinvention according to any one of the above claims wherein the oleandrincrosses the blood brain barrier (BBB) after administration to thesubject. 26) The invention according to any one of the above claimswherein the oleandrin has a clearance rate for brain tissue of nogreater than 4 L/hr. 27) The invention according to any one of the aboveclaims wherein the composition excludes neriifolin. 28) The inventionaccording to any one of the above claims wherein oleandrin providesimproved clinical benefit over neriifolin administered at an equivalentdose. 29) The invention according to any one of the above claims,wherein oleandrin is the sole active agent present in the composition.